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Regulated membrane localization of Tiam1, mediated by the NH2-terminal pleckstrin homology domain, is required for Rac-dependent membrane ruffling and C-Jun NH2-terminal kinase activation.

Michiels F, Stam JC, Hordijk PL, van der Kammen RA, Ruuls-Van Stalle L, Feltkamp CA, Collard JG - J. Cell Biol. (1997)

Bottom Line: Similar to V12Rac1, Tiam1 stimulates the activity of the c-Jun NH2-terminal kinase (JNK).This Rac-dependent stimulation of JNK also requires membrane association of Tiam1.We conclude that the regulated membrane localization of Tiam1 through its NH2-terminal PH domain determines the activation of distinct Rac-mediated signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: The Netherlands Cancer Institute, Antoni van Leeuwenhoekhuis, Division of Cell Biology, Amsterdam.

ABSTRACT
Rho-like GTPases, including Cdc42, Rac, and Rho, regulate signaling pathways that control actin cytoskeletal structures and transcriptional activation. The Tiam1 gene encodes an activator of Rac1, and similarly to constitutively activated (V12)Rac1, overexpression of Tiam1 in fibroblasts induces the formation of membrane ruffles. Tiam1 contains a Dbl homology (DH) domain and adjacent pleckstrin homology (PH) domain, hallmarks for activators of Rho-like GTPases. Unique for Tiam1 are an additional PH domain and a Discs-large homology region in the NH2-terminal part of the protein. Here we show that both in fibroblasts and COS cells, membrane localization of Tiam1 is required for the induction of membrane ruffling. A detailed mutational analysis, in combination with confocal laser scanning microscopy and immunoelectron microscopy, demonstrates that the NH2-terminal PH domain of Tiam1, but not the DH-adjacent PH domain, is essential for membrane association. This NH2-terminal PH domain of Tiam1 can be functionally replaced by the myristoylated membrane localization domain of c-Src, indicating that the primary function of this PH domain is to localize the protein at the membrane. After serum starvation, both membrane association of Tiam1 and ruffling can be induced by serum, suggesting that receptor stimulation induces membrane translocation of Tiam1. Similar to V12Rac1, Tiam1 stimulates the activity of the c-Jun NH2-terminal kinase (JNK). This Rac-dependent stimulation of JNK also requires membrane association of Tiam1. We conclude that the regulated membrane localization of Tiam1 through its NH2-terminal PH domain determines the activation of distinct Rac-mediated signaling pathways.

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Serum-dependent localization of Tiam1. Confocal images of NIH3T3 cells expressing C1199 Tiam1 (A and B) or MS-C580  Tiam1 (C) proteins. Cells were serum starved for 24 h (A and C), followed by stimulation with serum for 2 h (B). In the merged pictures,  Tiam1 proteins are shown in green and F-actin in red. These optical sections were taken at the basal sites of the cells and reveal the presence of stress fibers in serum-starved NIH3T3 cells. Magnification is 300×. (D and E) Immuno-EM localization of C1199 Tiam1 after  serum starvation (D) or after readdition of serum for 2 h (E). In F, the distribution of gold particles in at least 40 cross sections of cells  were scored and presented as the percentage of cross sections with gold particles being predominantly at the membrane (M) or in the  cytoplasm (C). Bars: (A–C) 40 μm; (D and E) 400 nm.
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Figure 8: Serum-dependent localization of Tiam1. Confocal images of NIH3T3 cells expressing C1199 Tiam1 (A and B) or MS-C580 Tiam1 (C) proteins. Cells were serum starved for 24 h (A and C), followed by stimulation with serum for 2 h (B). In the merged pictures, Tiam1 proteins are shown in green and F-actin in red. These optical sections were taken at the basal sites of the cells and reveal the presence of stress fibers in serum-starved NIH3T3 cells. Magnification is 300×. (D and E) Immuno-EM localization of C1199 Tiam1 after serum starvation (D) or after readdition of serum for 2 h (E). In F, the distribution of gold particles in at least 40 cross sections of cells were scored and presented as the percentage of cross sections with gold particles being predominantly at the membrane (M) or in the cytoplasm (C). Bars: (A–C) 40 μm; (D and E) 400 nm.

Mentions: PH domains have been identified in other signaling molecules as protein–protein and/or protein–phospholipid interaction motifs that are required for the controlled targeting of these proteins to the plasma membrane (Lemmon et al., 1996). In tissue sections of skin and certain carcinomas, endogenous Tiam1 is predominantly present in the cytoplasm. Also in some cell lines, where we can envision endogenous Tiam1 by Western blotting, including T-lymphoma cells and neuronal cells, it is mainly localized in the cytoplasmic fraction (data not shown), suggesting that Tiam1 may translocate to the plasma membrane after receptor stimulation. So far, however, we have not been able to identify a receptor-mediated signaling pathway involving Tiam1 activation. To investigate whether membrane translocation of exogenous Tiam1 could be visualized in NIH3T3 cells, we analyzed whether serum could affect the localization and capacity of Tiam1 to induce membrane ruffling. As shown in Fig. 8 A, NIH3T3 cells transiently expressing the C1199 Tiam1 protein showed no membrane ruffling after serum starvation for 24 h. Almost no Tiam1 protein was present at the plasma membrane, and F-actin was mostly concentrated in lamellipodia in the Tiam1-expressing cells. Since these optical sections were taken at the basal site of the cells to illustrate the lamellipodia, stress fibers are also visible. Note that after serum starvation for 24 h, NIH3T3 cells still contain some stress fibers, in contrast to Swiss 3T3 cells. Immuno-EM confirmed that most of the cells contained significantly less Tiam1 at the plasma membrane after serum starvation (Fig. 8, D and F). The residual membrane-associated Tiam1 might be sufficient for the presence of lamellipodia under these conditions. Addition of serum induced membrane localization and subsequent ruffling of C1199 Tiam1-expressing cells after 2 h (Fig. 8, B and E), at which time point seruminduced stress fibers have already decreased. A quantification of these results is presented in Fig. 8 F. Similar results were obtained with FL1591 Tiam1, C1199-ΔPHc, and C1199ΔDHR Tiam1 (data not shown). In contrast, after 24 h of serum starvation the MS-C580 Tiam1 protein remained at the plasma membrane and still induced membrane ruffling (Fig. 8 C). This suggests that the serum-induced membrane translocation of Tiam1 is mediated by the NH2-terminal PH domain.


Regulated membrane localization of Tiam1, mediated by the NH2-terminal pleckstrin homology domain, is required for Rac-dependent membrane ruffling and C-Jun NH2-terminal kinase activation.

Michiels F, Stam JC, Hordijk PL, van der Kammen RA, Ruuls-Van Stalle L, Feltkamp CA, Collard JG - J. Cell Biol. (1997)

Serum-dependent localization of Tiam1. Confocal images of NIH3T3 cells expressing C1199 Tiam1 (A and B) or MS-C580  Tiam1 (C) proteins. Cells were serum starved for 24 h (A and C), followed by stimulation with serum for 2 h (B). In the merged pictures,  Tiam1 proteins are shown in green and F-actin in red. These optical sections were taken at the basal sites of the cells and reveal the presence of stress fibers in serum-starved NIH3T3 cells. Magnification is 300×. (D and E) Immuno-EM localization of C1199 Tiam1 after  serum starvation (D) or after readdition of serum for 2 h (E). In F, the distribution of gold particles in at least 40 cross sections of cells  were scored and presented as the percentage of cross sections with gold particles being predominantly at the membrane (M) or in the  cytoplasm (C). Bars: (A–C) 40 μm; (D and E) 400 nm.
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Figure 8: Serum-dependent localization of Tiam1. Confocal images of NIH3T3 cells expressing C1199 Tiam1 (A and B) or MS-C580 Tiam1 (C) proteins. Cells were serum starved for 24 h (A and C), followed by stimulation with serum for 2 h (B). In the merged pictures, Tiam1 proteins are shown in green and F-actin in red. These optical sections were taken at the basal sites of the cells and reveal the presence of stress fibers in serum-starved NIH3T3 cells. Magnification is 300×. (D and E) Immuno-EM localization of C1199 Tiam1 after serum starvation (D) or after readdition of serum for 2 h (E). In F, the distribution of gold particles in at least 40 cross sections of cells were scored and presented as the percentage of cross sections with gold particles being predominantly at the membrane (M) or in the cytoplasm (C). Bars: (A–C) 40 μm; (D and E) 400 nm.
Mentions: PH domains have been identified in other signaling molecules as protein–protein and/or protein–phospholipid interaction motifs that are required for the controlled targeting of these proteins to the plasma membrane (Lemmon et al., 1996). In tissue sections of skin and certain carcinomas, endogenous Tiam1 is predominantly present in the cytoplasm. Also in some cell lines, where we can envision endogenous Tiam1 by Western blotting, including T-lymphoma cells and neuronal cells, it is mainly localized in the cytoplasmic fraction (data not shown), suggesting that Tiam1 may translocate to the plasma membrane after receptor stimulation. So far, however, we have not been able to identify a receptor-mediated signaling pathway involving Tiam1 activation. To investigate whether membrane translocation of exogenous Tiam1 could be visualized in NIH3T3 cells, we analyzed whether serum could affect the localization and capacity of Tiam1 to induce membrane ruffling. As shown in Fig. 8 A, NIH3T3 cells transiently expressing the C1199 Tiam1 protein showed no membrane ruffling after serum starvation for 24 h. Almost no Tiam1 protein was present at the plasma membrane, and F-actin was mostly concentrated in lamellipodia in the Tiam1-expressing cells. Since these optical sections were taken at the basal site of the cells to illustrate the lamellipodia, stress fibers are also visible. Note that after serum starvation for 24 h, NIH3T3 cells still contain some stress fibers, in contrast to Swiss 3T3 cells. Immuno-EM confirmed that most of the cells contained significantly less Tiam1 at the plasma membrane after serum starvation (Fig. 8, D and F). The residual membrane-associated Tiam1 might be sufficient for the presence of lamellipodia under these conditions. Addition of serum induced membrane localization and subsequent ruffling of C1199 Tiam1-expressing cells after 2 h (Fig. 8, B and E), at which time point seruminduced stress fibers have already decreased. A quantification of these results is presented in Fig. 8 F. Similar results were obtained with FL1591 Tiam1, C1199-ΔPHc, and C1199ΔDHR Tiam1 (data not shown). In contrast, after 24 h of serum starvation the MS-C580 Tiam1 protein remained at the plasma membrane and still induced membrane ruffling (Fig. 8 C). This suggests that the serum-induced membrane translocation of Tiam1 is mediated by the NH2-terminal PH domain.

Bottom Line: Similar to V12Rac1, Tiam1 stimulates the activity of the c-Jun NH2-terminal kinase (JNK).This Rac-dependent stimulation of JNK also requires membrane association of Tiam1.We conclude that the regulated membrane localization of Tiam1 through its NH2-terminal PH domain determines the activation of distinct Rac-mediated signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: The Netherlands Cancer Institute, Antoni van Leeuwenhoekhuis, Division of Cell Biology, Amsterdam.

ABSTRACT
Rho-like GTPases, including Cdc42, Rac, and Rho, regulate signaling pathways that control actin cytoskeletal structures and transcriptional activation. The Tiam1 gene encodes an activator of Rac1, and similarly to constitutively activated (V12)Rac1, overexpression of Tiam1 in fibroblasts induces the formation of membrane ruffles. Tiam1 contains a Dbl homology (DH) domain and adjacent pleckstrin homology (PH) domain, hallmarks for activators of Rho-like GTPases. Unique for Tiam1 are an additional PH domain and a Discs-large homology region in the NH2-terminal part of the protein. Here we show that both in fibroblasts and COS cells, membrane localization of Tiam1 is required for the induction of membrane ruffling. A detailed mutational analysis, in combination with confocal laser scanning microscopy and immunoelectron microscopy, demonstrates that the NH2-terminal PH domain of Tiam1, but not the DH-adjacent PH domain, is essential for membrane association. This NH2-terminal PH domain of Tiam1 can be functionally replaced by the myristoylated membrane localization domain of c-Src, indicating that the primary function of this PH domain is to localize the protein at the membrane. After serum starvation, both membrane association of Tiam1 and ruffling can be induced by serum, suggesting that receptor stimulation induces membrane translocation of Tiam1. Similar to V12Rac1, Tiam1 stimulates the activity of the c-Jun NH2-terminal kinase (JNK). This Rac-dependent stimulation of JNK also requires membrane association of Tiam1. We conclude that the regulated membrane localization of Tiam1 through its NH2-terminal PH domain determines the activation of distinct Rac-mediated signaling pathways.

Show MeSH
Related in: MedlinePlus